Guanosine tetra‐ and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA‐based fluorescent sensors for the live‐cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp‐specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell‐to‐cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA‐based sensors can be broadly adapted to study bacterial stringent response.
Live‐Cell Imaging of Guanosine Tetra‐ and Pentaphosphate (p)ppGpp with RNA‐based Fluorescent Sensors**
Guanosine tetra‐ and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA‐based fluorescent sensors for the live‐cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp‐specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell‐to‐cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA‐based sensors can be broadly adapted to study bacterial stringent response.
more » « less- NSF-PAR ID:
- 10306370
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie
- Volume:
- 133
- Issue:
- 45
- ISSN:
- 0044-8249
- Page Range / eLocation ID:
- p. 24272-24276
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
null (Ed.)Abstract Background Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus , c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr ). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability. Results We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp. Conclusions In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus .more » « less
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null (Ed.)ABSTRACT The stringent response involves accumulation of (p)ppGpp, and it ensures that survival is prioritized. Production of (p)ppGpp requires purine synthesis, and upregulation of an operon that encodes the purine salvage enzyme xanthine dehydrogenase (Xdh) has been observed during stringent response in some bacterial species, where direct binding of ppGpp to a TetR-family transcription factor is responsible for increased xdh gene expression. We show here that the plant pathogen Ralstonia solanacearum has a regulatory system in which the LysR-family transcription factor XanR controls expression of the xan operon; this operon encodes Xdh as well as other enzymes involved in purine salvage, which favor accumulation of xanthine. XanR bound upstream of the xan operon, a binding that was attenuated on addition of either ppGpp or cyclic di-guanosine monophosphate (c-di-GMP). Using a reporter in which enhanced green fluorescent protein (EGFP) is expressed under control of a modified xan promoter, XanR was shown to repress EGFP production. Our data suggest that R. solanacearum features a regulatory mechanism in which expression of genes encoding purine salvage enzymes is controlled by a transcription factor that belongs to a different protein family, yet performs similar regulatory functions.more » « less
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Stress and starvation causes bacterial cells to activate the stringent response. This results in down-regulation of energy-requiring processes related to growth, as well as an upregulation of genes associated with survival and stress responses. Guanosine tetra- and pentaphosphates (collectively referred to as (p)ppGpp) are critical for this process. In Gram-positive bacteria, a main function of (p)ppGpp is to limit cellular levels of GTP, one consequence of which is reduced transcription of genes that require GTP as the initiating nucleotide, such as rRNA genes. In Streptomycetes, the stringent response is also linked to complex morphological differentiation and to production of secondary metabolites, including antibiotics. These processes are also influenced by the second messenger c-di-GMP. Since GTP is a substrate for both (p)ppGpp and c-di-GMP, a finely tuned regulation of cellular GTP levels is required to ensure adequate synthesis of these guanosine derivatives. Here, we discuss mechanisms that operate to control guanosine metabolism and how they impinge on the production of antibiotics in Streptomyces species.more » « less
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